Horseradish peroxidase (HRP) is a heme enzyme activated by peroxides. The reactive oxidized forms produced by peroxides are: Compound I and II, containing the heme group in the oxo-ferryl π-cation and oxo-ferryl forms, respectively. These high valent HRP intermediates can use peroxides to be converted to the native form (Fe³⁺) or can abstract electrons from other reducing agents such as aldehydes and phenols. The return of HRP to the native form complete the catalytic cycle of the enzyme. In this work, the regeneration of native HRP from HRP Compound II generated by hydrogen peroxide was analyzed in the presence of methylene (MB⁺) in the dark and under irradiation with a 500 Watts halogen lamp. In the dark rate of HRP compound II reduction was similar to that determined in the absence of MB⁺, suggesting that hydrogen peroxide was the reducing species. Under irradiation, the reduction of HRP Compound II was accompanied by UV-visible spectrometry in the presence of different methylene blue (MB⁺) concentrations over the range of 0.45 to 27.5 mM. The plot of the rate of the Compound II reduction to the native form in function of MB⁺ concentration revealed a quadratic hyperbolic profile. The plot of 1/rate in function of 1/[MB⁺] exhibited two slopes indicating that two reducing species are involved in the process. The increase of MB⁺ concentration favors the formation of the dimer form of the dye. In this form, the triplet MB⁺ (³MB^*) abstracts one electron of its partner generating the methylene blue neutral radical (MB^·). Considering that the experiments were done in aerated medium in which the ³MB^* should be significantly quenched by molecular oxygen, we postulate that by increasing the dye concentration, HRP Compound II was reduced preferentially by H₂O₂ or by MB^·, respectively. The HRP Compound II reduction was investigated over the pH range from 3.0 to 9.0. In the dark, the highest rate for HRP Compound II reduction was attained at pH = 4.0 and under irradiation, the optimal pH was nine. The subtraction of the pH curve obtained under irradiation from that obtained in the dark results in a pH curve very similar to the pH curve of ³MB^* quantum yield. From these data it was observed that MB^· contributes significantly to the Compound II reduction above pH = 6.0 but the peroxide contribution, although decreasing, remains significant above this pH value. This result corroborates that, in this system, H₂O₂ and MB^· are the electron donating species.